Newly developed reconstituted high-density lipoprotein containing sphingosine-1-phosphate induces endothelial tube formation

doi:10.1016/j.atherosclerosis.2006.10.020

Atherosclerosis

Volume 194, Issue 1, September 2007, Pages 159-168

https://doi.org/10.1016/j.atherosclerosis.2006.10.020 Get rights and content

Abstract

Reconstituted high-density lipoprotein (rHDL) has been shown to produce a rapid regression of atherosclerosis in animal models and humans. Sphingosine-1-phosphate (S1P), which is a bioactive lipid in HDL, plays a role in mitogenesis, endothelial cell motility, and cell survival, as well as organization and differentiation into a vessel. In this study, we examined the direct role of a newly developed rHDL, [POPC(1-palmitoyl-2-oleoyl phosphatidylcholine)/S1P/apolipoproteinA–I(A–I)]rHDL containing S1P in tube formation in endothelial cells (ECs) as well as cholesterol efflux in macrophage. The effect of (POPC/S1P/A–I)rHDL on cholesterol efflux in macrophage was similar to that of conventional rHDL, (POPC/A–I)rHDL. In addition, (POPC/S1P/A–I)rHDL induced EC proliferation through the activation of phospho-Akt and phospho-extracellular-signal-regulated kinases (p-ERK) 1/2 and EC tube formation, and this effect was blocked by inhibitors of Akt, ERK and endothelial nitric-oxide synthase (eNOS). In addition, (POPC/S1P/A–I)rHDL-induced p-ERK1/2 activation and EC tube formation can be mainly attributed to S1P-stimutated signaling through S1P₂ and S1P₃ as determined by an anti-sense strategy. In conclusion, (POPC/S1P/A–I)rHDL induces cholesterol efflux independently of S1P but has additional S1P-mediated effects on EC tube formation mediated by Akt/ERK/NO through S1P₂ and S1P₃. In the future, these new discs may be useful for the treatment of atherosclerotic and ischemic cardiovascular disease, such as acute coronary syndrome and atherosclerosis obliterans.

Introduction

High-density lipoproteins (HDL) are a heterogeneous group of small, dense lipoproteins. A low HDL level is one of the strongest predictors of coronary risk [1]. The negative correlation between coronary artery disease (CAD) and plasma HDL-cholesterol has been attributed to the ability of HDL to take up cellular cholesterol from the periphery and to mediate the transport of excess cholesterol to the liver [2].

HDL plays a crucial role in reverse-cholesterol transport [3]. A key factor in this process is the interaction between lipid depleted of apolipoproteinA–I (A–I) and the transmembrane protein ABCA1, which results in cellular cholesterol efflux. In addition, HDL has pleiotropic effects, in that it promotes anti-inflammation, including inhibition of the expression of adhesion molecules and monocyte chemotaxis, and anti-coagulation [4]. Although HDL is a target in the treatment of atherosclerotic CAD [5], there are currently only a limited number of therapeutic options to promote HDL. However, several exciting therapeutic strategies have recently been developed and are currently the focus of intense research such as the infusion of A–I [6], [7] or reconstituted(r)HDL, which may act as cholesterol acceptors [5]. rHDL that mimic the structure of A–I have anti-atherogenic effects in animal models [8], [9], [10]. A–I mimetic peptides have also been shown to inhibit inflammation [11] and thrombus formation [12]. In addition, Nissen et al. reported that 45 patients suffering from acute coronary syndrome received weekly infusions of A–I-Milano-phospholipid complexes over a period of 5 weeks. In patients who received the infusions, there was a significant regression in plaque size compared with baseline using intracoronary vascular ultrasound. Despite the small number of patients involved, this study has focused considerable attention on A–I-infusion as a therapeutic concept [13], and more attention has been paid to rHDL for the treatment of coronary atherosclerosis.

Sphingosine-1-phosphate (S1P), which is contained in HDL, is known to exhibit a wide variety of biological activities in many mammalian and other vertebrate cell types. S1P induces cell survival signaling and is involved in migration, proliferation, angiogenesis [14] and cytoskeletal changes in endothelial cells (ECs) mediated by a specific family of G protein-coupled (GPCRs) sphingosine 1-phosphate receptors [S1P₁ (formerly endothelial differentiation gene-1 [Edg-1]), S1P₂ (Edg-5), S1P₃ (Edg-3), S1P₄ (Edg-6) and S1P₅ (Edg-8)] [15]. Therefore, we developed new rHDL, [(POPC(1-palmitoyl-2-oleoyl phosphatidylcholine)/S1P/A–I)]rHDL containing S1P. The new rHDL may induce cholesterol efflux and may have additional effects for cell survival, including proliferation and angiogenesis. In the future, the rHDL may be useful for the treatment of atherosclerotic cardiovascular disease, such as acute coronary syndrome and atherosclerosis obliterans.

Section snippets

Materials

The following antibodies and reagents were purchased: A–I (Calbiochem); PD98059, 2-(2′-amino-3′-methoxyphenyl) oxanaphthalen-4-one, a specific inhibitor of extracellular-signal-regulated kinases (ERK) (PD98059) (New England BioLabs); S1P and l-NAME (Nω-Nitro-l-arginine methyl ester hydrochloride) (Sigma); Akt inhibitor (1L-6-Hydroxymethyl-chio-inositol-2-(R)-2-O-methyl-3-O-octadecylcarbonate)(Merck); antibodies for Akt, phospho(p)-Akt, ERK1/2 and p-ERK1/2 (Thr²⁰²/Tyr²⁰⁴) (Cell Signaling

Characterization of discoidal rHDL

Characterization of discoidal rHDL by non-denaturing gradient gel electrophoresis showed that most of the (POPC/A–I)rHDL were 10.0 nm in diameter (Fig. 1). Minor populations of particles 10.7, 12.1. and 16.0 nm in diameter were also apparent in this preparation. The (POPC/S1P/A–I)rHDL particles were 9.8 nm in diameter. These rHDL particles did not contain discrete populations of larger particles. The absence of lipid-free apoA–I in the discoidal rHDL-containing tracks indicates that apoA–I was

Discussion

Previous studies have shown that rHDL has anti-atherosclerotic effects through the improvement of cholesterol efflux in addition to pleiotropic effects. In this study, we developed new rHDL and examined the mechanisms of their influence on these pleiotropic effects. We investigated the impact of ERK activation on new rHDL-mediated cell signaling and the induction of HCEC tube formation. Our results also identified ERK and Akt activation through S1P₂ and S1P₃ as a key player in HCEC tube

Study limitation

In this study, we assessed whether newly developed rHDL induced tube formation in an in vitro study. More detailed investigations for the clinical effects will be required to determine if rHDL induces the development of tube formation in vivo study because S1Ps may be diluted by other lipoproteins in plasma.

In summary, we have demonstrated a novel function of newly developed rHDL, in that HCEC tube formation was mediated by a ERK/Akt/NO pathway through S1P₂ and S1P₃ as well as enhanced

Acknowledgments

This work was supported by funds from the Central Research Institute of Fukuoka University, Japan. KAR is a Principal Research Fellow of the National Heart Foundation of Australia. We are grateful to Dr. K. Hirano (Osaka University) for providing cDNA of SR-BI.

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Newly developed reconstituted high-density lipoprotein containing sphingosine-1-phosphate induces endothelial tube formation

Abstract

Introduction

Section snippets

Materials

Characterization of discoidal rHDL

Discussion

Study limitation

Acknowledgments

Atherosclerosis

J Lipid Res

Biochem Biophys Res Comm

Biochim Biophys Acta

Cell

J Lipid Res

Meth Enzymol

Clin Chim Acta

J Biol Chem

J Biol Chem

J Biol Chem

Mol Cell

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

Meth Enzymol

Eur J Cancer

High-density lipoprotein—the clinical implications of recent studies

N Engl J Med